The study of protein/protein interaction, as exemplified, e.g., by the identification of ligands for receptors, is an area of great interest. Even when a ligand or ligands for a given receptor are known, there is interest in identifying more effective or more selective ligands. G-protein coupled receptors, GPCRs, also known as seven transmembrane receptors (7TMR), will be discussed herein as a non-exclusive example of a class of proteins which can be characterized in this way. However, any proteins that interact, for example, members of a metabolic pathway or a cascade, are suitable for use with the instant assay.
GPCRs are the largest class of cell surface receptors known for humans and thus are considered a prime application of the invention. Ligands that modulate signaling by GPCRs include hormones, neurotransmitters, peptides, glycoproteins, lipids, nucleotides, and ions. GPCRs also are known to be sense receptors, e.g., receptors for light, odor, a pheromone, and taste. Given these diverse and numerous roles, GPCRs are the subject of intense research, for example, for chemical defense and bio-defense applications and for drugs useful in treating various conditions. Many drug discovery successes have already occurred. For example, Howard, et al., Trends Pharmacol. Sci., 22:132 140 (2001) has estimated that over 50% of marketed drugs act on such receptors.
“GPCRs” as used herein, refer to any member of the GPCR superfamily of receptors. This superfamily is characterized by a seven-transmembrane domain (7TM) structure. Examples of these receptors include, but are not limited to, the class A or “rhodopsin-like” receptors; the class B or “secretin-like” receptors; the class C or “metabotropic glutamate-like” receptors; the Frizzled and Smoothened-related receptors; the adhesion receptor family or EGF-7TM/LNB-7TM receptors; adiponectin receptors and related receptors; and chemosensory receptors including odorant, taste, vomeronasal and pheromone receptors. As examples, the GPCR superfamily in humans includes, but is not limited to, receptor molecules described by Vassilatis, et al., Proc. Natl. Acad. Sci. USA, 100:4903 4908 (2003); Takeda, et al., FEBS Letters, 520:97 101 (2002); Fredricksson, et al., Mol. Pharmacol., 63:1256 1272 (2003); Glusman, et al., Genome Res., 11:685 702 (2001); and Zozulya, et al., Genome Biol., 2:0018.1 0018.12 (2001).
In brief, the general mechanism of action of GPCR function is as follows: 1) a GPCR binds a ligand 2) causing a conformational change thereby 3) stimulating a cascade of cellular events that lead to a change in cell physiology. GPCRs transduce signals by modulating activity of a plurality of intracellular proteins, such as, heterotrimeric guanine nucleotide binding proteins (G proteins) and β arrestins. In the case of G proteins, the ligand-receptor complex stimulates guanine nucleotide exchange and dissociation of the G protein heterotrimer into α and βγ subunits. In other circumstances, a β arrestin can substitute for a G protein, oppose G protein signaling, synergize G protein signaling and so on.
Both the GTP-bound a subunit and the βγ heterodimer have been observed to regulate various cellular effector proteins, including adenylyl cyclase and phospholipase C (PLC). In conventional cell-based assays for GPCRs, receptor activity is monitored by measuring the output of a G protein-regulated effector pathway, such as, accumulation of cAMP, produced by adenylyl cyclase; or release of intracellular calcium, e.g., stimulated by PLC activity.
Conventional G protein-based signal transduction assays have been difficult to develop for some targets for a variety of reasons. For example, first, different GPCRs are coupled to different G protein-regulated signal transduction pathways. Traditional G protein-based assays are dependent on knowing the G protein specificity of the target receptor, or the assays require engineering of the cellular system to force couple the target receptor to a selected G protein effector pathway. Second, since the GPCR superfamily is so large, all cells express many endogenous GPCRs (as well as other receptors and signaling factors). Thus, the measured effector pathways can be modulated by endogenous molecules in addition to the target GPCR. This phenomenon can cause false positive or false negative results, e.g., when attempting to identify selective modulators of a target GPCR.
Regulation of G protein activity is not the only result of ligand/GPCR binding. See, for example, Luttrell, et al., J. Cell Sci., 115:455 465 (2002), and Ferguson, Pharmacol. Rev., 53:1 24 (2001), which review activities that can lead to attenuation or termination of the GPCR signal. These termination processes are useful to prevent excessive cell stimulation, and to enforce a temporal linkage between an extracellular signal and the corresponding intracellular pathway.
In general, binding of an agonist to a GPCR causes serine and threonine residues at the C terminus of the receptor molecule to be phosphorylated by GPCR kinase. Agonist-complexed C terminal-phosphorylated GPCRs then interact with arrestin family members, e.g., α arrestin, β arrestin or β arrestin 2, which down modulate or arrest receptor signaling. The binding can inhibit coupling of the receptor to G proteins, thereby targeting the receptor for internalization, followed by degradation and/or recycling. For example, binding of an arrestin, such as β arrestin 2 to a phosphorylated GPCR can reduce activity of the target GPCR in different ways. The simplest mechanism for an arrestin to inhibit activation of its target is to bind to the intracellular domain of the GPCR thereby blocking the binding site for the heterotrimeric G protein and preventing extracellular signals from activating the pathway (desensitization). Another regulatory mechanism employed by arrestins is linkage of the receptor to elements of the membrane internalization machinery (e.g., clathrin-mediated endocytosis) which initiates internalization of the receptor in a coated vesicle for fusion with an endosome. Once at an endosome, the receptor can be either targeted for degradation (e.g., by lysosomes) or can be recycled to the plasma membrane where it can once again be activated.
Hence, the binding of a ligand to a GPCR can be said to “modulate” the interaction between the GPCR and arrestin proteins, since the binding of ligand to GPCR causes the arrestin to bind to the GPCR, thereby modulating its activity. Herein, when “modulates” or any form thereof is used with respect to interaction or binding, it refers simply to some change in the way the two proteins of the invention interact, when, for example, a test compound or ligand is present, as compared to how these two proteins interact, in its absence. Hence, modulate includes mere binding of two molecules. For example, the presence of the test compound may strengthen or enhance the interaction of the two proteins, weaken it, block it, inhibit it, redirect it, lessen it or modify it in some way, manner or form which is detectable, or the test compound may facilitate the likelihood of interaction and so on.
In some circumstances, 7TMR signaling can occur independent of G proteins. Thus, on 7TMR binding of ligand, β arrestin instead of G protein is recruited to precipitate or to initiate a signaling cascade in the cell. See, for example, Violin & Lefkowitz, Trends Pharm Sciences 28(8)416-422, 2007 and DeFea, Br J Pharm 1-12, doi:10.1038/sj.bjp.0707508, 2007 who summarize the two independent and interdependent signaling pathways beginning at the activated 7TMR, and which can involve both a G protein and a β arrestin; or involve either a G protein or a β arrestin.
Thus, for example, known antagonists of a 7TMR activate β arrestin signaling. Propranolol, a known antagonist of the β2 adrenergic receptor (ADRB2) and of G protein signaling, was found to be a partial agonist of β arrestin signaling, activating β arrestin-initiated pathways, as observed practicing the instant invention.
Cell signaling events responsive to extracellular stimuli are generally mediated by protein-protein interactions. Protein-protein interactions therefore are of great interest to cell physiologists. One tool to monitor these interactions involves using a split or permuted reporter activating protein, such as, tobacco etch virus (TEV) protease. The split portions of the protease regain activity when co-expressed as a fusion construct with interacting proteins. Wehr, et al., “Monitoring Regulated Protein-protein Interactions Using Split TEV”, Nature Methods, 3:985-993 (2006). This property has been used in conjunction with transcription-coupled reporter systems.
This understanding has led to alternate methods for assaying activation and inhibition of GPCRs. One of these methods involves monitoring interaction with arrestins in an intact cell carrying transcription machinery. An advantage of this approach is that no knowledge of G protein pathways is necessary. See, e.g., U.S. Pat. No. 7,049,076: “Method for Assaying Protein-Protein Interaction” to Lee at al. Lee et al. teach a reporter system that requires transcription-coupled reporter systems. According to Lee et al., a peptidic transcription factor is cleaved from a first protein when two proteins interact. The second protein is a transcription factor that activates a reporter gene. The factor then accomplishes the reporter function by transport to the nucleus to effect transcription of a detectable reporter. Because the method is dependent on transcription, the method is inoperable, for example, in platelets, artificial packages or units, such as liposomes, cochleates, virus-like particles, and viral particles.
Oakley, et al., Assay Drug Dev. Technol., 1:21 30 (2002) and U.S. Pat. Nos. 5,891,646 and 6,110,693, “Methods Of Assaying Receptor Activity and Constructs Useful in Such Methods” to Barak et al., describe assays where the redistribution of fluorescently-labeled arrestin molecules in the cytoplasm to activated receptors on the cell surface is measured. These methods rely on high resolution imaging of cells to measure arrestin relocalization and receptor activation. It is recognized by the skilled artisan that this is a complex, involved procedure that can be waylaid by the affinity and interaction of the complementary enzyme fragments used therein which can compete with the desired modulator-induced interaction. Hence, the method suffers from false positives arising from an auto-reassociation of the enzyme, independent of ligand binding. A simpler, more robust assay with a lower incidence of false positives, and which is more readily adaptable to high throughput screening would be desirable.
Various other US patents and patent applications dealing with these points have issued and have been filed. For example, U.S. Pat. No. 6,528,271, “Inhibition Of β-Arrestin Mediated Effects Prolongs and Potentiates Opioid Receptor-Mediated Analgesia” to Bohn et al., features assays to screen for pain-controlling medications, where inhibition of β arrestin binding is measured. Published U.S. patent applications, such as 2004/0002119, 2003/0157553 and 2003/0143626; and U.S. Pat. No. 6,884,870, describe different forms of assays involving GPCRs. U.S. Pat. No. 7,128,915 features similar GPCR technology. U.S. Pat. No. 7,049,076 mentioned above generally featuring GPCR activities or screening assays demonstrate the importance of GPCR research.
Thus, one feature of the present invention, i.e., providing a simpler assay for monitoring and/or determining modulation of specific protein/protein interactions, for example, receptor-mediated physiology, such as GPCR-mediated cellular responses, where the proteins include, but are not limited to, membrane-bound proteins, including receptors in general, and GPCRs as an important example, is satisfactory for addressing a desired need in the art.